The goal of this research project is to investigate the distribution of muscle proteins and calcium-sequestering vesicles in mitotic spindles, cleavage furrows, and pseudopodial regions of motile cells. The ultimate goal is to determine how actin and myosin function in these regions of cells. Actin, myosin, tubulin, tropomyosin and paramyosin will be localized by fluorescent antibodies and by fluorescent tracer techniques developed by the applicant. Distribution of the proteins will be examined on both the light and electron microscope level. To minimize problems of translocation into mitotic spindles, both intranuclear and cytoplasmic spindles will be examined. The problems of regulation of actin-myosin interactions in cell motility will be approached by looking for a vesicular system capable of actively concentrating calcium in regions where cell-movements occur. Pilot experiments have demonstrated that there is an extensive vesicular system containing deposits of calcium that is associated with mitotic spindles, cleavage furrows and with the pseudopods of macrophages. ATP-dependent calcium localization methods will be used to determine whether the system is an active calcium pump like the sarcoplasmic reticulum system of muscle. The localization of calcium-binding vesicles with respect to the contractile proteins in the spindle apparatus, in the cleavage furrow and in other motile areas of non-muscle cells will be investigated. The techniques necessary for this work are -1) protein isolation, 2) antibody preparation, 3) tissue culturing, and 4) microscopy; fluorescence, phase, polarization and electron.